N-糖酰胺酶F(PNGase F)在大肠杆菌中的表达纯化及其脱糖基化作用鉴定

N-糖酰胺酶F(PNGase F)是由革兰氏阴性菌——脑膜炎脓杆菌分泌的一种分子量为34.8 kDa的去糖基化酶.本研究通过PCR技术扩增出945 bp的N-糖酰胺酶F基因,经酶切、连接,构建了大肠杆菌表达载体pET28a-PNGase F.将pET28a-PNGase F转入大肠杆菌BL21中,16℃诱导表达及Ni柱纯化后,SDS-PAGE鉴定,获得超过95%纯度的可溶性表达的N-糖酰胺酶F.对Na+依赖的中性氨基酸转运蛋白1(SNAT1)和2(SNAT2)进行脱糖基化作用检测,结果证明:纯化的N-糖酰胺酶F对SNAT1和SNAT2具有高效的脱糖基化作用.

Expression and purification of PNGase F( PNGase F) in escherichia coli and its functional study

N-glycosidase F（PNGase F）is a enzyme of deglycosylation,secreted by the gram-negative bacterium Flavobacterium meningosepticum.It’s theoretical molecular weight is about 34.8 kDa.In this study,the PNGase F gene of 945 bp was amplified by PCR technique.A prokaryotic expression vector,pET28a-PNGase F,was constructed by digestion and ligation.After E.coli（BL21）containing pET28a-PNGase F plasmids was cultured and induced by IPTG at 16 ℃,soluble PNGase F was purified through Ni column,and the purity is up to over 95% by SDS-PAGE identification.Deglycosylation of purified PNGase F on neutral amino acid transporter SNAT1 and SNAT2 showed that PNGase F is highly effective and both SNAT1 and SNAT2 contain glycosylated sites.